Volume 135, Issue 4, Pages 1267-1276 (October 2008) Role of Ghrelin in the Relationship Between Hyperphagia and Accelerated Gastric Emptying in Diabetic Mice  Pieter–Jan Verhulst, Betty De Smet, Inge Saels, Theo Thijs, Luc Ver Donck, Dieder Moechars, Theo L. Peeters, Inge Depoortere  Gastroenterology  Volume 135, Issue 4, Pages 1267-1276 (October 2008) DOI: 10.1053/j.gastro.2008.06.044 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Targeted disruption of the GHS-R1 gene. (A) Structure of the targeting vector, wild-type locus, targeted locus, and Cre-excised locus. Boxes representing the exons in open and shaded boxes are the noncoding and coding regions, respectively. Arrows indicate the position of the PCR primers used for genotyping the wild-type and targeted allele. (B) Expression of the GHS-R1 transcript in brain and pituitary of both genotypes. Results are means ± SEM (n = 6) relative expression levels after normalization to β-actin. Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Changes in blood glucose levels (A), body weight (B), and daily (24 hours) food intake (C) in STZ-induced diabetic GHS-R+/+ and GHS-R−/− mice as a function of time. Results are represented as the means ± SEM (n = 11–14 animals/group). *P < .05; **P < .01; ***P < .001 (GHS-R+/+ vs GHS-R−/− mice). Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Time-dependent effects of diabetes on plasma ghrelin levels (A) and hypothalamic mRNA expression of ghrelin (B); GHS-R (C); and the neuropeptides AgRP (D), NPY (E), and POMC (F) in ad libitum fed (10 am) GHS-R+/+ and GHS-R−/− mice before and 7, 15, and 27 days after the induction of diabetes. Results are represented as the means ± SEM (n = 3–7 animals/group). *P < .05; **P < .01; ***P < .001 (GHS-R+/+ vs GHS-R−/− mice). Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Time-dependent changes in gastric half-excretion time (Thalf) (A) and lag time (Tlag) (B) as determined by the [13C]octanoic acid breath test in diabetic GHS-R+/+ and GHS-R−/− mice. Results are means ± SEM (n = 11–14 animals/group). †P < .01 (GHS-R+/+, compared with day 0); ‡P < .05; ‡‡P < .01; ‡‡‡P < .001 (GHS-R−/−, compared with day 0). Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Electrical field stimulation of strips from nondiabetic GHS-R+/+ and GHS-R−/− mice resulted in on-relaxations (A) and off-contractions (B). Results are means ± SEM (n = 3 animals/group; n = 3 strips/animal). *P < .05 (GHS-R+/+ vs GHS-R−/− mice). On-relaxations are expressed as a percentage of the maximal relaxation obtained after stimulation with 10−5 mol/L nitroglycerin; off-contractions are expressed in grams/square millimeters. Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Effect of STZ-induced diabetes on the neural responses of fundic strips from wild-type and mutant mice. Representative tracings of EFS-induced responses in nondiabetic and diabetic GHS-R+/+ (A) and GHS-R−/− (B) mice are shown. The graphs summarize the change in tension of the on-relaxations (C and D) and off-contractions (E and F) in GHS-R+/+ (left) and GHS-R−/− (right) mice. Results are represented as the means ± SEM (n = 3–5 animals/group; n = 3 strips/animal). *P < .05; **P < . 01; ***P < .001 (nondiabetic vs diabetic mice). Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Pharmacologic characterization of neural responses of nondiabetic wild-type mice (A and B). (A) Representative tracings obtained in the presence of L-NAME (3 × 10−4 mol/L), and after cumulative addition of atropine (5 × 10−6 mol/L), SR140333 (5 × 10−7 mol/L), SR48968 (5 × 10−7 mol/L), and tetrodotoxin (TTX) (3 × 10−4 mol/L), respectively. (B) Tension of the off-contractions obtained with the various pharmacologic treatments. Results are mean ± SEM (n = 3 animals/group; n= 3 strips/animal). *P < .05; ** P < .001. Effect of diabetes on the contractile response to ACh and substance P in fundic strips (C and D). Dose-response curves to ACh (C) and substance P (D) in strips from nondiabetic (open symbols) and diabetic (solid symbols) GHS-R+/+ and GHS-R−/− mice. Results are represented as the means ± SEM (n = 3–5 animals/group; n = 3 strips/animal). Gastroenterology 2008 135, 1267-1276DOI: (10.1053/j.gastro.2008.06.044) Copyright © 2008 AGA Institute Terms and Conditions